Ammonia metabolism is critical for normal health. Inappropriate ammonia metabolism in the kidney leads to metabolic acidosis and in the liver leads to ammonia encephalopathy. In the central nervous system increased extracellular ammonia alters neuronal function and can lead to encephalopathy. Accordingly, understanding the cellular and molecular mechanisms of ammonia metabolism, which includes ammonia transport, is important. Recent studies have identified a novel family of ammonium ion (NH4+)-specific transporters. These proteins were first identified in yeast and in plants, and homologues are present throughout nature. In model systems, such as yeast, plants and bacteria, these are intrinsic membrane proteins that mediate high-affinity, ammonium-specific transport and whose expression is physiologically regulated. Two of these proteins, RhBG and RhCG are expressed in the connecting segment and the collecting duct of the kidney, and exhibit polarized expression. These observations lead us to postulate that RhBG and RhCG are integral membrane, physiologically-regulated ammonium-ion transporters that play critical roles in renal ammonia metabolism. The broad, long-term objectives of this project are to define the roles of RhBG and RhCG in mammalian renal physiology. To do so, the Specific Aims of the current proposal are to: (1) Define the regulation of mouse renal RhBG and RhCG expression and vesicular trafficking in response to specific clinical conditions associated with altered renal ammonia metabolism; (2) Determine the mechanism of extracellular ammonia-stimulated changes in RhBG- and RhCG-mediated ion transport; and, (3) identify the specific ion-transport characteristics of RhBG and RhCG. We will utilize in vivo animal models of altered renal ammonia metabolism, metabolic acidosis and alkalosis and hypokalemia, to define the regulation of RhBG and RhCG expression and cellular localization, a cultured collecting duct cell line, mIMCD-3, for in vitro studies examining the cellular mechanisms underlying regulation of RhBG and RhCG-mediated transport, and heterologous expression systems in which to define the specific ion transport characteristics of RhBG and RhCG.